Lysyl oxidase (LO) plays a critical role in the formation and repair of the extracellular matrix (ECM) by oxidizing lysine residues in elastin and collagen, thereby initiating the formation of covalent crosslinkages which stabilize these fibrous proteins. Its catalytic activity depends upon both its copper cofactor and a unique carbonyl cofactor and has been shown to extend to a variety of basic globular proteins, including histone H1. Although the three-dimensional structure of LO has yet to be determined, the present treatise offers hypotheses based upon its primary sequence, which may underlie the prominent electrostatic component of its unusual substrate specificity as well as the catalysis-suppressing function of the propeptide domain of prolysyl oxidase. Recent studies have demonstrated that LO appears to function within the cell in a manner, which strongly modifies cellular activity. Newly discovered LO-like proteins also likely play unique roles in biology.

Lysyl oxidase (EC 1.4.3.13) oxidizes peptidyl lysine to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent crosslinkages that insolubilize these extracellular proteins. Recent findings raise the possibility that this enzyme may also function intracellularly. The present study provides evidence by immunocytochemical confocal microscopy, Western blot analysis, enzyme assays, and chemical analyses for lysyl oxidase reaction products that this enzyme is present and active within rat vascular smooth muscle cell nuclei. Confocal microscopy indicates its presence within nuclei of 3T3 fibroblasts, as well.Lysyl oxidase (LO; EC 1.4.3.13) catalyzes the post-translational modification of elastin and collagen, by oxidizing selected lysine residues within these proteins to peptidyl ␣-aminoadipic-␦-semialdehyde. Subsequent spontaneous reactions of the peptidyl aldehydes yield covalent cross-linkages (1). LO is synthesized as a 46-kDa preproenzyme by fibrogenic cells. After signal peptide cleavage and N-glycosylation, the resulting 50-kDa N-glycosylated proenzyme is secreted (2) and proteolytically processed in the extracellular space to a mature enzyme of 31 Ϯ 1 kDa (3).Although initiation of the cross-linking of elastin and collagen is a critical function of LO, there is evidence that it may have additional biological roles. The mature enzyme isolated from bovine aorta is chemotactic for monocytes and lymphocytes in assays in vitro, with the chemotactic effect requiring a functional active site (4). Moreover, LO expression is negligibly low in several neoplastically transformed cell lines (5), including fibroblasts transformed with the Ha-ras oncogene (6, 7). It is of particular interest in this regard that a murine ras recision gene, the expression of which appears to suppress the tumorigenic effect of Ha-ras, encodes LO (6-9). The basis of this apparent effect of LO has yet to be understood. However, a recent report notes that transfection of revertants derived from ras-transformed NIH 3T3 cells with LO antisense but not LO sense constructs induced a change in the relatively ''loose'' chromatin packing state of the revertants to the tighter chromatin packing state of the original transformants (10). These results raise the possibility that LO may directly or indirectly exert effects on nuclear components. In the present study, we provide evidence that LO occurs and catalytically functions within the nuclei of fibrogenic cells. EXPERIMENTAL PROCEDURESCell Culture. Neonatal rat aorta smooth muscle cells (NRASMCs), explanted from 2-to 3-day-old rat pups as described (11, 12), were used in first passage at or just prior to confluency. Swiss 3T3 fibroblasts (American Type Culture Collection) were used at confluency after 3-4 days of culture in passages 9-12. Cells were cultured in DMEM containing 3.7 g of NaHCO 3 per l, 100 units of penicillin per ml, 100 g of streptomycin per ml, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate (GIBCO͞BRL), and 10% fetal bovine serum ...

LPS-induced TNFα factor (LITAF) is a multiple functional molecule whose sequence is identical to small integral membrane protein of the lysosome/late endosome (SIMPLE). LITAF was initially identified as a transcription factor that activates transcription of proinflammatory cytokine in macrophages in response to LPS. Mutations of the LITAF gene are associated with a genetic disease, called Charcot-Marie-Tooth syndrome. Recently we have reported that mRNA levels of LITAF and tumor necrosis factor superfamily member 15 (TNFSF15) are upregulated by AMPK. The present study further assesses their biological functions. Thus, we show that AICAR, a pharmacological activator of AMPK, increases the abundance of LITAF and TNFSF15 in the LNCaP and C4-2 prostate cancer cells, which is abrogated by shRNA or dominant negative mutant of AMPK α1 subunit. Our data further demonstrate that AMPK activation upregulates the transcription of LITAF. Intriguingly, silencing LITAF by shRNA enhances proliferation, anchorage-independent growth of these cancer cells, and tumor growth in xenograft model. In addition, our study reveals that LITAF mediates the effect of AMPK by binding to a specific sequence in the promoter region. Furthermore, we show that TNFSF15 remarkably inhibits the growth of prostate cancer cells and bovine aortic endothelial cells in vitro with a more potent effect toward the latter. In conjuncture, intratumor injection of TNFSF15 significantly reduces the size of tumors and number of blood vessels and induces changes characteristic of tumor cell differentiation. Therefore, our studies for the first time establish the regulatory axis of AMPK-LITAF-TNFSF15. They also suggest that LITAF may function as a tumor suppressor.

Lysyl oxidase (LO), an enzyme secreted by vascular smooth muscle cells (VSMC), initiates the covalent crosslinking of polypeptide chains within collagen and elastin. The present study reveals that purified LO strongly induces directional migration of VSMC in an in vitro assay system. LO-dependent chemotaxis, but not chemokinesis, was abolished by beta-aminopropionitrile, an active site inhibitor of LO, or by catalase, as well as by prior heat denaturation. This indicates that the H(2)O(2) product of amine oxidation by LO is critical to the expression of its chemotactic activity. The results indicate that the chemotactic response requires direct access between LO and a substrate molecule (or molecules) tightly associated with the VSMC. The addition of LO to VSMC elevated the levels of intracellular H(2)O(2), enhanced stress fiber formation, and focal adhesion assembly, is consistent with the induction of the chemotactic response.

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